Many in vitro experiments with enzymes are performed in dilute conditions that do not resemble the cellular environment. Water, small metabolites, nucleic acids, and other proteins in the surrounding impact an enzyme's structure, intrinsic dynamics, and function. To account for these macromolecular crowding effects, synthetic polymers are often used to simulate the intracellular environment as it exists in vivo, to determine the impact volume exclusion and soft interactions have on the conformation and function of the enzyme. These synthetic crowders have been found to impact substrate binding and catalytic efficiency. However, the precise impact of these synthetic crowders cannot be known from the kinetic assay alone. To explore the crowder's role in enzyme functions at the molecular level, a fluorescence spectroscopy-based study was designed that utilized the intrinsic tryptophan (TRP) fluorescence of E. coli prolyl-tRNA (Ec ProRS) synthetase to probe catalytically important conformational changes as well as thermal denaturation of the protein. Experiments involving three variants of Ec ProRS, each containing TRP in only one of the three domains of the protein, are used to probe individual domain conformation under crowding conditions. Thermal denaturation of Ec ProRS, probed with fluorescence spectroscopy in the presence of monomeric and polymeric crowders demonstrate the differing effects on protein stability. Lastly, fluorescence resonance energy transfer (FRET) is used to measure the change in the dynamics of the insertion domain under crowded conditions. These experiments ultimately show the environmentally induced conformational changes of enzymes, which help researchers design structure-based drugs or antibiotics. Preliminary results of the study will be presented.

Studying the effects of crowding on structure and conformational changes of Prolyl-tRNA synthetase using fluorescence spectroscopy

ACS Spring 2022

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